Throughout this specification, including any claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and any appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
Anthracyclines: Doxorubicin
The anthracycline doxorubicin has been in clinical use for several decades, and is still among the most widely used chemotherapeutic agents for treatment of a variety of neoplasms (see, e.g., Weiss, 1992; Zagotto et al., 2001; Lothstein et al., 2001).
Doxorubicin has several cytotoxic actions. It binds to DNA and inhibits both DNA and RNA synthesis, but its main cytotoxic action appears to be mediated through an effect on topoisomerase II (a DNA gyrase), the activity of which is markedly increased in proliferating cells. The significance of the enzyme lies in the fact that during replication of the DNA helix, reversible swivelling needs to take place around the replication fork in order to prevent the daughter DNA molecule becoming inextricably entangled during mitotic segregation. The swivel is produced by topoisomerase II, which nicks both DNA strands and subsequently reseals the breaks. Doxorubicin intercalates in the DNA and its effect is, in essence, to stabilise the DNA-topoisomerase II complex after the strands have been nicked, thus causing the process to seize up at this point.
Liposomal Delivery
Despite many years of research in developing new and better anthracyclines, little or no change in the molecular structure of doxorubicin made it to the clinics. However, with the development of liposomal formulations, its delivery form underwent a major improvement (see, e.g., Tardi et al., 1996; Gabizon, 2001). Compared to systemic application of doxorubicin in its free form, liposomal doxorubicin exhibits significant advantages, as for example reduced acute and chronic toxicities. Improved loading procedures, resulting in high doxorubicin packing efficiencies, further increased the therapeutic index of encapsulated doxorubicin (see, e.g., Horowitz et al., 1992; Haran et al., 1993). Another major step forward was the development of polyethyleneglycol (PEG)-coated liposomes. This coating prevents opsonization and reduces the uptake by macrophages from the reticulo-endothelial system, in turn resulting in prolonged circulation times, as compared to free doxorubicin or to non-coated liposomes (see, e.g., Vaage et al., 1992; Robert and Gianni, 1993; Gabizon et al., 1996; Uster et al., 1996). PEG-liposome-encapsulated doxorubicin (commercially available as Caelyx® and Doxil®) is now in routine clinical use, and innovations such as the coupling of targeting-enhancing features (e.g., tumor cell specific antibodies or ligands) will further enhance its therapeutic value (see, e.g., Park et al., 2002; Pan et al., 2003; Koning et al., 1999; Koning et al., 2003).
Both Caelyx® and Doxil® consist of: doxorubicin hydrochloride (2 mg/mL); N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE) (3.19 mg/mL); fully hydrogenated soy phosphatidylcholine (HSPC) (9.58 mg/mL); cholesterol (3.19 mg/mL); ammonium sulfate (˜2 mg/mL); histidine (as a buffer); hydrochloric acid and/or sodium hydroxide (for pH control); sucrose (to maintain isotonicity); and water-for-injection.
The endothelial lining of healthy blood vessels effectively prevents escape of liposomes from the circulation. In contrast, angiogenesis-associated vascular abnormalities of many solid tumors, do allow extravasation of long-circulating PEG-liposomes into the tumor stroma (see, e.g., Yuan et al., 1994). Despite this tumor-specific accumulation, liposomes are, however, not taken up by tumor cells. Instead, doxorubicin is gradually released into the interstitial space (see, e.g., Horowitz et al., 1992; Harasym et al., 1997). Given the intracellular localization of its molecular targets, sufficient cellular uptake of doxorubicin is required for its action (see, e.g., Speth et al., 1988; Lothstein et al., 2001). However, since doxorubicin does not possess the optimal degree of lipophilicity for efficient plasma membrane traversal, this might be a limiting factor for its efficacy (see, e.g., Heijn et al., 1999; Washington et al., 2001).
Improved Liposomal Delivery
The inventors have demonstrated that the cellular uptake of free doxorubicin, and with that its cytotoxic action, is greatly enhanced by co-administration of the short-chain sphingolipid analogue N-hexanoyl-sphingomyelin (referred to herein as “C6-SM”). Due to a truncated acyl-chain (attached to the amino group at the second carbon position of sphingosine), this lipid spontaneously inserts into lipid bilayers, and exchanges easily between membranes (see, e.g., Jeckel and Wieland, 1993; Ghidoni et al., 1999).
Despite the promising results obtained in vitro, several problems are to be expected when doxorubicin and the lipid analogue are co-administered separately, without any physical (e.g., liposomal) binding, in vivo, including: (1) For intravenous application of lipid solutions toxic solvents, such as ethanol, are required. (2) Lipids typically show poor plasma solubility, and might form undesired deposits on vessel walls. (3) Due to their high affinity for serum components, such as albumin and apolipoproteins, large amounts of lipid might be needed to obtain the desired effect. (4) Doxorubicin and the lipid analogue will most likely exhibit differences in biodistribution, and will thus not be delivered at the same site at the same time, which is a prerequisite for the drug-uptake enhancing effect.
The inventors have also demonstrated in vitro that liposomal anthracycline (for example, doxorubicin) which has been enriched with glycosphingolipid (for example, N-octanoyl-glucosylceramide, referred to herein as “C8-GlcCer”) greatly enhances drug transfer to tumor cells, in turn leading to an increased cytotoxicity. Furthermore, these glycosphingolipids have doxorubicin uptake-enhancing properties comparable to that of C6-SM, but with a significantly lower toxicity. Incorporation of these glycosphingolipids into the liposome bilayer would effectively circumvent any solubility-related problems. Advantages of co-delivery of doxorubicin and the lipid analogue (within the same liposomal complex) include avoidance of lipid-solubility related toxicities and of differences in biodistribution. In addition, the steric barrier provided by PEG (for example, in PEG-liposome-encapsulated doxorubicin) would reduce any interaction of the glycosphingolipids with serum components. Such liposomes ensure co-delivery of the anthracycline (e.g., doxorubicin) and glycosphingolipid at the same site. Additionally, this effect was found to be fully reproducible in the presence of high serum concentrations, strongly supporting the feasibility of in vivo applications. These results were fully reproducible when N-octanoyl-glucosylceramide was post-inserted into Caelyx®, a commercial liposomal doxorubicin preparation. Taken together, these results demonstrate that glycosphingolipids-enrichment is a major improvement of a well established doxorubicin formulation.
The improved formulation offers many advantages. For example, the improved drug uptake permits the use of formulations with lower drug (e.g., amphiphilic drug) (e.g., anthracycline) (e.g., doxorubicin) content to achieve the same result, thereby reducing undesired side effects, e.g., myocardial toxicity.
Liotta et al., 1999 describes the use of certain sphingolipids, including certain glycosphingolipids (see, e.g., compounds listed from page 25 onwards therein) as active agents (e.g., drugs) in the treatment of abnormal cell proliferation. Although combination therapy is discussed (see, e.g., pages 72-73 therein), anthracyclines and doxorubicin are mentioned only as part of a long list of possible drugs. Although pharmaceutical compositions are discussed (see, e.g., starting on page 107 therein), liposomal suspensions and liposome formulations are mentioned (see, e.g., page 110 therein) only as part of a long list of possible formulations. In view of this document, and from among the myriad of other embodiments therein, both theoretical and exemplified, the skilled reader would not seriously contemplate pharmaceutical formulations comprising certain short-chain sphingolipids, as described herein, and drugs (e.g., anthracyclines), let alone corresponding liposomal formulations. Nowhere is there any teaching or suggestion of the use of short-chain sphingolipids as an adjunct to improved the efficacy of drugs (e.g., anthracyclines). Furthermore, the substantial improvement offered by such formulations (and demonstrated by the inventors) is both surprising and unexpected.